【作者】 王蕾；

【导师】 杨向东； 黎健；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2006， 硕士

【摘要】 研究背景：尼克酰胺腺嘌呤二核苷酸磷酸氧化酶(nicotinamide adenine dinucleotide phosphate oxidase，NADPH氧化酶，NOX)是由多个亚基组成的酶复合体，包含胞膜成分细胞色素b558(gp91phox和p22phox)和胞浆成分p47phox、p67phox及Rac(小的GTP结合蛋白)五个亚组分。gp91phox(有NADPH、heme、FAD的潜在结合位点)和p22phox是NADPH氧化酶的酶促核心，它们受胞浆成分p47phox、p67phox及Rac的调节。近几年的研究发现，在各种组织中存在着5种NOX的同功酶，分别称为NOX1、NOX2、NOX3、NOX4、NOX5，其中NOX2和NOX4均在血管内皮细胞中表达，但NOX4表达水平比NOX2高20倍。进一步的研究表明，血管内皮细胞产生的活性氧(reactive oxygen species，ROS)主要来源于NOX4。我们的研究证实，高浓度低密度脂蛋白(low density lipoprotein，LDL)通过上调NOX4的表达和增加ROS的产生引起内皮细胞凋亡，提示由高浓度LDL引起的氧化应激在动脉粥样硬化的发病机制中起重要作用。令人感兴趣的是，拮抗动脉粥样硬化的保护性因素高密度脂蛋白(high-density lipoprotein，HDL)和降脂的他汀类药物近年来已被证实具有抗氧化的作用，但它们的抗氧化机制尚不清楚。本研究首先用HDL及其主要载脂蛋白apoAI和辛伐他汀预处理人脐静脉内皮细胞(human umbilical vein endothelial cells，HUVEC)，再加入高浓度LDL观察NOX各亚基表达及ROS生成的变化，探讨HDL和辛伐他汀的抗氧化机制。 目的：分析HUVEC中NOX各亚基的表达；分析HDL及其主要载脂蛋白apoAI与辛伐他汀对HUVEC内NOX4、NOX2、p67phox、p22phox和Rac表达及ROS生成的影响，研究HDL、apoAI和辛伐他汀对LDL的拮抗作用。更多还原

【Abstract】 Background: Nicotinamide adenine dinucleotide phosphate oxidase(NADPH oxidase) is a multicomponent enzyme complex, which consists of 5 major subunits: a plasma membrance spanning cytochrome b558 composed of a large subunit gp91phox and a smaller p22phox subunit and 3 cytosolic components p47phox,p67phox and Rac.Upon stimulation,the three cytosolic components translocate to the membrance and assemble with cytochrome b558,resulting in enzyme activation and burst production of ROS.ROS of human vascular system mostly derive from NADPH oxidase.Gp91phox was firstly found in phagocyte cells.and the novel gp91phox homologues of NOX families such as NOX1, N0X3, N0X4, N0X5 have been found in different organs (colon, kidney, spleen) and vascular smooth muscle cells in recent years. Latest studies indicated that N0X4 gene may be important in the ROS generation in endothelial cells. Several cell types in the artery wall have been show to oxidise lipoprotein, including endothelial cells, smooth muscle cells, which proceed the progression of atherosclerosis. In the recent years, the antioxidant activity of high density lipoprotein (HDL) to directly impede some of processes in atherogenesis has receiving increasing attention. But the mechanism of HDL is not clear involving chelate of transition metal ions, a reservoir for lipid peroxides, HDL-associated enzymes. In these studies, we observed the change of N0X4, N0X2, p47phox, p67phox, p22phox and Rac expression and intracellular ROS level in HUVEC stimulated by HDL, simvastadin or apoAI before low density lipoprotein(LDL) addition.Objectives: To investigate the mRNA expression of p47phox, p67phox, p22phox and Rac gene in HUVEC; then to elucidate the change of N0X4, N0X2, p67phox, p22phox and Rac expression in HUVEC stimulated by HDL, simvastadin or apoAI before LDL addition.Methods: U937 cell was used as the positive control of mRNA expression of p47phox, p67phox, p22phox and Rac. N0X4, N0X2, p67phox, p22phox and Rac expression in HUVEC was measured by RT-PCR. N0X4 and N0X2 expression in HUVEC was更多还原